Differential expression and release of exosomal miRNAs by human islets under inflammatory and hypoxic stress
Pancreatic islets produce non-coding microRNAs (miRNAs) that regulate islet cell function and survival. Our earlier investigations revealed that human islets undergo significant damage due to various types of stresses following transplantation and release miRNAs. Here, we sought to identify and validate exosomal miRNAs (exo-miRNAs) produced by human islets under conditions of cellular stress, preceding loss of cell function and death. We also aimed to identify islet stress signalling pathways targeted by exo-miRNAs to elucidate potential regulatory roles in islet cell stress.
Human islets were subjected to proinflammatory cytokine and hypoxic cell stress and miRNA from exosomes was isolated for RNA sequencing and analysis. Stress-induced exo-miRNAs were evaluated for kinetics of expression and release by intact islets for up to 48 h exposure to cytokines and hypoxia. A subset of stress-induced exo-miRNAs were assessed for recovery and detection as biomarkers of islet cell stress in a diabetic nude mouse xenotransplant model and in patients undergoing total pancreatectomy with islet auto-transplantation (TPIAT). Genes and signalling pathways targeted by stress-induced exo-miRNAs were identified by Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and direct interactions of miRNAs with downstream signalling targets were validated in human islet cells using the miRNA Tests for Read Analysis and Prediction (MirTrap) system.
Global exo-miRNA sequencing revealed that 879 miRNA species were released from human islets and 190 islet exo-miRNAs were differentially expressed in response to proinflammatory cytokines, hypoxia or both. Release of exo-miRNAs hsa-miR-29b-3p and hsa-miR-216a-5p was detected within 6 h of exposure to cytokines and hypoxia. The remaining subset of stress-induced exo-miRNAs, including hsa-miR-148a-3p and islet cell damage marker hsa-miR-375, showed delayed release at 24–48 h, correlating with apoptosis and cell death. Stress and damage exo-miRNAs were significantly elevated in the circulation in human-to-mouse xenotransplant models and in human transplant recipients. Elevated blood exo-miRNAs negatively correlated with post-transplant islet function based on comparisons of stress and damage exo-miRNA indices with Secretory Unit of Islet Transplant Objects (SUITO) indices. KEGG analysis and further validation of exo-miRNA targets by MirTrap analysis revealed significant enrichment of islet mRNAs involved in phosphoinositide 3-kinase/Akt and mitogen-activated protein kinase signalling pathways.
The study identifies exo-miRNAs differentially expressed and released by islets in response to damage and stress. These exo-miRNAs could serve as potential biomarkers for assessing islet damage and predicting outcomes in islet transplantation. Notably, exo-miRNAs 29b-3p and 216a-5p could be detected in islets prior to damage-released miRNAs and indicators of cellular apoptosis and death. Thus, these stress-induced exo-miRNAs may have potential diagnostic value for detecting early islet stress prior to progressive loss of islet cell mass and function. Further investigations are warranted to investigate the utility of these exo-miRNAs as early indicators of islet cell stress during prediabetic conditions.
KeywordsCellular stress Exosomal miRNA Islet cell damage Islet transplantation PI3K–Akt signalling pathway
BCL2-associated death promoter
CCAAT-enhancer-binding protein homologous protein
Damage miRNA index
Forkhead box protein O1
Hypoxia inducible factor 1α
Heat-shock protein 70
Hypoxia (experimental treatment)
Kyoto Encyclopedia of Genes and Genomes
miRNA Tests for Read Analysis and Prediction
Mammalian target of rapamycin
Nanoparticle tracking analysis
Stress miRNA index
Secretory Unit of Islet Transplant Objects
Transmission electron microscopy
Total pancreatectomy with islet auto-transplantation
X-box binding protein 1 isoform
Technical support by Y. Liu and A. Rahman, Islet Cell Laboratory, Baylor Simmons Transplant Institute, is acknowledged.
PBS designed the study, performed experiments, analysed data and wrote the manuscript. SV analysed data and wrote and revised the manuscript. GY and CMD assisted in performing in vivo experiments, data analysis and manuscript preparation. XW and JG analysed microRNA sequencing data and assisted in manuscript preparation. MCL and BN designed and supervised the study, analysed data and wrote the manuscript. All authors approve the final version of the manuscript. BN is responsible for the integrity of the work as a whole.
The study was supported by an institutional grant from Baylor University Medical Center.
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
- 16.Coronel MM, Geusz R, Stabler CL (2017) Mitigating hypoxic stress on pancreatic islets via in situ oxygen generating biomaterial. Biomaterials 129:139–151. https://doi.org/10.1016/j.biomaterials.2017.03.018 Google Scholar
- 50.Tuo Y, Xiang M (2018) mTOR: a double-edged sword for diabetes. J Leukoc Biol. https://doi.org/10.1002/jlb.3mr0317-095rr
- 51.Chang CA, Akinbobuyi B, Quintana JM, Yoshimatsu G, Naziruddin B, Kane RR (2018) Ex-vivo generation of drug-eluting islets improves transplant outcomes by inhibiting TLR4-mediated NFkB upregulation. Biomaterials 159:13–24. https://doi.org/10.1016/j.biomaterials.2017.12.020 Google Scholar